Proteins, acting directly or through their enzymatic functions, contribute in major proportion to many diseases in animals and man. Classical therapeutic methods have generally focused on modulating protein function with exogenous compounds that interact with proteins, with the goal of moderating their disease-causing or disease-potentiating functions. Recently, however, attempts have been made to moderate the actual production of certain proteins using molecules that direct protein synthesis, such as intracellular RNA. By interfering with the production of proteins, it has been hoped to effect therapeutic results with maximal desired effect and minimal side effects. It is the general object of such therapeutic approaches to interfere with, or otherwise modulate, the expression of genes that lead to undesired protein formation.
One method for inhibiting specific gene expression is the use of oligonucleotides and oligonucleotide analogs as “antisense” agents. Antisense technology involves directing oligonucleotides, or analogs thereof, to a specific, target messenger RNA (mRNA) sequence, whereby transcription is modulated. Thus, antisense technology permits modulation of essential functions of intracellular nucleic acids.
As antisense oligonucleotides and oligonucleotide analogs are now accepted as therapeutic agents holding great promise for therapeutic and diagnostic methods, it has become desirable to produce them in relatively large quantities. In some applications, it is necessary to produce large numbers of small batches of diverse oligonucleotides or their analogs for screening purposes. In other cases, for example in the production of therapeutic quantities of oligonucleotides and their analogs, it is necessary to make large batches of the same oligonucleotide, or analog thereof.
Three principal methods have been used for the synthesis of oligonucleotides. The phosphotriester method, as described by Reese, Tetrahedron 1978, 34, 3143; the phosphoramidite method, as described by Beaucage, in Methods in Molecular Biology: Protocols for Oligonucleotides and Analogs; Agrawal, ed.; Humana Press: Totowa, 1993, Vol. 20, 33–61; and the H-phosphonate method, as described by Froehler in Methods in Molecular Biology: Protocols for Oligonucleotides and Analogs Agrawal, ed.; Humana Press: Totowa, 1993, Vol. 20, 63–80. Of these three methods, the phosphoramidite method has become a defacto standard in the industry.
In order to meet in the increasing demand for oligonucleotides and their analogs, it increasingly necessary to produce the intermediates for oligonucleotide synthesis in greater quantity, and with satisfactory purity, to satisfy the increased demand for the final products. In the case of phosphoramidite synthesis, it is desirable to scale up the process of making phosphoramidite precursors. As impurities in phosphoramidite precursors will impact oligonucleotide product purity, the phosphoramidite must be of exceptional purity. However, the classical methods of purifying phosphoramidites, involving purification on silica gel columns, are less than suitable for scale up. Larger scale purification requires the use of larger silica gel columns, which in turn results in increased residence time on the silica gel columns, and increased volumes of mobile phase solvent. As phosphoramidites tend to degrade in a time-wise manner on silica gels, large volume silica gel columns mean greater proportional loss of the desired product and a concomitant increase in undesirable contaminants. One of the results of scale-up of phosphoramidite purification on silica gels is thus a decrease in percent product yield, which tends to offset any advantages of scale that have been realized at other steps in the synthesis.
Another result of scale-up is an increase in time required to remove solvent from the product. As column volume increases, diffusion causes an increase in fraction volume, a large portion of which is simply mobile phase solvent. While solvent stripping may be a minor consideration in small-scale purification, it can require substantial amounts of time as scale increases.
Of course, increased column volume, and the resulting increase in product fraction volume, result in greater expenditures of operator time.
There is thus a need for a scalable, economic process for purifying phosphoramidites that avoids the problems of phosphoramidite degradation and solvent usage associated with conventional silica gel column purification.